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Biomedical subjects

K Watabe

Publications and source records attributed to K Watabe.

At least 109 records · Page 6Linked to original sources
PubMed1990

Transfection and stable transformation of adult mouse Schwann cells with SV-40 large T antigen gene.

Cultured Schwann cells derived from adult mouse dorsal root ganglia and peripheral nerves were transfected with a plasmid containing SV-40 large T antigen gene, and 25 colonies of stable transformants were obtained, one of which was expanded and recloned. This transfected cell line, designated MS1, expressed SV-40 large T antigen and showed continuous cell growth with a doubling time of 27 hours. The MS1 cells had distinct Schwann cell phenotypes such as S-100 protein, laminin, 2',3'-cyclic nucleotide 3'-phosphodiesterase and P0 protein, as shown by immunofluorescence microscopy. When MS1 cells were exposed to dibutyryl cyclic adenosine-3',5'-monophosphate (dbc AMP), they extended long bipolar processes two- to ten-fold longer than those of untreated MS1 cells and frequently formed whorl-like alignments similar to palisade formations or organoid patterns observed in human Schwannomas and neurofibromas. These results suggest that transformed Schwann cells can be a useful model for analyzing regulatory mechanisms of Schwann cells, neuron-Schwann cell interactions and experimental Schwann cell neoplasms in vitro.

Animals↗
PubMed1990

Antisense oligodeoxyribonucleotides inhibit the expression of the gene for hepatitis B virus surface antigen.

The effect of a series of antisense oligodeoxyribonucleotide [oligo(dN)] on the expression of the surface antigen (HBsAg) gene of human hepatitis B virus (HBV) was examined using hepatocellular carcinoma cells that contain integrated HBV genomes. Of a number of antisense oligo(dN)s tested, synthetic 15-mers directed at the cap site of mRNA and regions of the translational initiation site of the HBsAg gene were found to be highly effective and inhibited viral gene expression by as much as 96%. The inhibition was specific to the HBsAg gene and appeared to be at the level of translation. These results suggest a therapeutic potential for antisense oligo(dN) in the treatment of patients who are chronically infected with HBV.

Base Sequence↗
PubMed1989

Human fetal Schwann cells in culture: phenotypic expressions and proliferative capability.

In this report we examined the phenotypic expressions and the proliferative capability of cultured human fetal Schwann cells. Antigens that were expressed included laminin, nerve growth factor receptor, neural cell adhesion molecule, S-100 protein, and that recognized by the monoclonal antibody HNK-1. In addition, HLA-A,B,C and HLA-DR, respectively, class I and class II antigens of the major histocompatibility complex, were demonstrated on Schwann cells. Mitotic capability was high, with an average of 34% of Schwann cells undergoing proliferation over a 2-day period.

Antigens, Surface↗
PubMed1989

Transplantation of human sympathetic neurons and adrenal chromaffin cells into parkinsonian monkeys: no reversal of clinical symptoms.

Cultured human fetal sympathetic ganglion explants or adrenal chromaffin cell aggregates were implanted into the left striatum of monkeys whose left nigrostriatal pathway had been lesioned with the neurotoxin MPTP. There was no clinical reversal of parkinsonian symptoms and PET scans did not show increased striatal fluorodopa uptake from pre-implant levels. At sacrifice, left striatal contents of dopamine were not statistically different from MPTP-treated but non-implanted controls. Histological examinations revealed pockets of extrinsic cells which were found at the end of needle tracks. There was no evidence of immune rejection. The extrinsic cells did not stain for tyrosine hydroxylase or neurofilament, suggesting that they were not dopaminergic neurons. The failure to reverse clinical parkinsonian symptoms highlights the stage of infancy of neural implantation in Parkinson's disease.

Adrenal Medulla↗
PubMed1989

Identification and nucleotide sequence of the minimal replicon of the low-copy-number plasmid pBS2.

The plasmid pBS2 has a low copy number and is endogenous to Bacillus subtilis. The replication of this plasmid depends on the function of most of the host's dna genes including dnaB, which is unique to B. subtilis and is required for both the initiation of chromosome replication and the DNA-membrane association. We have identified the region that is essential for the replication of pBS2 and determined the complete 2279-bp nucleotide sequence of this region. In this region, there are two stretches of sequence homologous to the 18-bp consensus sequence which commonly appears at the origin of replication of plasmids pUB110 and pC194. The entire region contains six sizable open reading frames. Two of them are probably translated. One open reading frame, designated ORF A, coding for 269 amino acids, has significant homology, in terms of amino acid sequence, with the open reading frame of the gene for the Rep U protein of plasmid pUB110. The similarities between pBS2 and other plasmids suggest that the pBS2 may also replicate as a rolling circle, which appears to be the salient feature of a mechanism of replication that is common to small plasmids in gram-positive bacteria.

Amino Acid Sequence↗
PubMed1989

Phagocytic activity of human adult astrocytes and oligodendrocytes in culture.

The phagocytic activity of human glial cells was examined in primary cultures obtained from normal human brain obtained at autopsy. Highly enriched cultures of astrocytes and oligodendrocytes were established using an enzyme digestion-Percoll density gradient method. These cultures were exposed to medium containing carbon particles (indian ink) for one-24 hours. Under phase contrast and immunofluorescence microscopy, carbon particles were demonstrated in the cytoplasm and processes of both astrocytes and oligodendrocytes. Electron microscopic examination revealed carbon particles phagocytized and segregated in these cells. These findings may support a view that both astrocytes and oligodendrocytes participate in phagocytosis in certain neurological diseases.

Astrocytes↗
PubMed1989

Human and simian glial cells infected by human T-lymphotropic virus type I in culture.

Although human T-lymphotropic virus type I (HTLV-I) has been implicated in the etiology of tropical spastic paraparesis (TSP) and HTLV-I associated myelopathy (HAM), the direct infectivity of the virus against constituent cells in the central nervous system remains undetermined. To investigate the neurotropism of HTLV-I, we exposed cultured human and simian glial cells to HTLV-I. Primary mixed glial cell cultures of astrocytes and oligodendrocytes were obtained from adult human and cynomolgus monkey (Macaca fascicularis) brains by an enzyme digestion-Percoll gradient method. After two weeks in vitro, the cells were co-cultured with irradiated MT-2 cells, an HTLV-I-producing T-cell line. Cultures were double stained with antibodies against cell-type specific markers and anti-HTLV-I p19 (gag) monoclonal antibody. The HTLV-I antigen was demonstrated in small numbers of glial fibrillary acidic protein-positive cells (astrocytes) and galactocerebroside-positive cells (oligodendrocytes) in both the human and simian cultures. Electron microscopy demonstrated the presence of type C virus-like particles in the cytoplasm of astrocytes. These results indicate that HTLV-I is capable of infecting human and simian glial cells in vitro.

Adult↗
PubMed1989

Surface hydrophobicity of spores of Bacillus spp.

The surface hydrophobicity of 12 strains of Bacillus spp. was examined in a hexadecane-aqueous partition system. Mature and germinated spores of Bacillus megaterium QM B1551 transferred to the hexadecane layer, while vegetative and sporulating cells did not. Wild-type spores were more hydrophobic than spores of an exosporium-deficient mutant of B. megaterium QM B1551, although the mutant spores were shown to be hydrophobic to some extent by using increased volumes of hexadecane. This result suggests that the exosporium is more hydrophobic than the spore coat and that the surface hydrophobicity of spores depends mainly on components of the exosporium. The surface hydrophobicity of spores of nine other species of Bacillus was also examined, and spores having an exosporium were more hydrophobic than those lacking an exosporium. Thus measurement of the hydrophobicity of spores by the hexadecane partition method may provide a simple and rapid preliminary means of determining the presence or absence of an exosporium.

Alkanes↗
PubMed1988

Binding of tissue-specific factors to the enhancer sequence of hepatitis B virus.

We have identified tissue-specific factors, in human hepatoma cells, that bind specifically to the transcriptional enhancer sequence of the human hepatitis B virus (HBV). Two different types of protein factor were found in nuclear extracts of hepatoma cells by gel mobility shift assay. One factor was observed in human hepatoma cells but not in human kidney, lung, or vein cells, or in embryonic mouse cells. The other was discovered in both human hepatoma cells and human vein cells. DNase I footprint analysis, using the enhancer fragment (164 bp, AccI-SphI) from HBV, revealed that two specific sites are recognized by the nuclear factors. These sites contain consensus octamer sequences which have been found in many other enhancer elements. These results strongly suggest that the two nuclear factors found in hepatoma cells play key roles in the function of the HBV enhancer.

Animals↗
PubMed1988

Synthesis of recombinant human single-chain urokinase-type plasminogen activator variants resistant to plasmin and thrombin.

Single-chain urokinase-type plasminogen activator (scu-PA), a potential therapeutic reagent for thrombosis, is activated in plasma by plasmin. The activated enzyme is further digested by plasmin to generate low-molecular-weight urokinase (LMW-UK), which has no affinity for fibrin. To circumvent this dual effect of plasmin, we synthesized in Escherichia coli a variant of scu-PA, which is not converted to LMW-UK on treatment with plasmin. In another variant, the activation cleavage site was modified such that activation by plasmin was slowed down and that inactivation by thrombin was greatly diminished. The combination of these variants may be applicable as an effective thrombolytic reagent for clinical use.

Drug Resistance↗
PubMed1988

Monoclonal antibodies to O4 antigen of Vibrio parahaemolyticus.

Four monoclonal antibodies were prepared against O4 antigen of Vibrio parahaemolyticus. All the antibodies were shown to be specific for O4 antigen by agglutination with heat-killed O-cells of the organism and precipitation with LPS preparations. The inhibition experiments of the precipitations with various sugars and oligosaccharides suggested that the combining sites of these hybridoma antibodies were directed to an antigenic determinant structure containing----3 and----6 linked D-glucose, D-galactose, and N-acetyl-D-galactosamine.

Agglutination↗
PubMed1987

Identification of the product of dnaB gene in Bacillus subtilis.

In order to detect the product of dnaB gene in B. subtilis, a gene which is involved in the initiation of DNA replication and the formation of the DNA-membrane complex, we synthesized an origopeptide of 15 amino acids which corresponds to a region near the carboxyl-terminal of the gene product, and raised antibody against the synthetic peptide. We have also employed a filter binding assay to measure the predicted DNA binding activity of the product of the dnaB gene, using the plasmid pUB110. The binding activity was detected after fractionation of cell lysates of B. subtilis on sucrose-density gradients. When the active fraction was prepared from a mutant which was temperature-sensitive for the dnaB gene, the DNA binding activity in the fraction showed significant thermolability. Furthermore, the binding activity was inhibited by the purified antibody raised against the synthetic peptide. These results suggest that the product of the dnaB gene does indeed have DNA binding activity, and that the filter binding assay and the antibody can be used for the detection and characterization of the gene product.

Amino Acid Sequence↗
PubMed1987

Characterization of a deletion mutant of bacteriophage phi 29.

A spontaneous deletion mutant of bacteriophage phi 29 (phi 29 delta 1) has been characterized. This mutant has a 1112-bp deletion, which covers almost the entire sequence of genes 14 and 15, including an early promoter (B2). While lysis is very delayed, the phage DNA synthesis and internal phage development appear to be normal in the cells infected with this deletion mutant. These results indicate that the early functions are intact in phi 29 delta 1. Our results also suggest that genes 14 and 15 are dispensable for bacteriophage phi 29 growth and that the B2 promoter may also be dispensable for early functions in phi 29.

Bacteriophages↗
PubMed1987

Effects of dnats genes on the replication of plasmids in Bacillus subtilis.

An essential region (2.3 kb) for the replication of a low-copy-number plasmid, pBS-2, has been identified and cloned into plasmid pHV60 in Bacillus subtilis. The resultant plasmid, pKW1, and two other plasmids, pC194 (medium copy number) and pTP5 (high copy number), were examined by double radio-labelling and gel electrophoresis to determine which host functions are required for their replication in B. subtilis. Replication of pKW1 requires the functions of most dna genes, in particular dnaB, C, E, F, G and H; pC194 requires only dnaG and H; and pTP5 requires dnaE, F, G and H. Thus dnaG and dnaH are required for the replication of all three plasmids tested, even though each plasmid showed a different spectrum of dependency on other host functions. Because of its greater dependence on host functions and its low copy number, pKW1 should be a useful model with which to investigate the function of host genes in the replication of DNA in B. subtilis. pKW1 should also be a useful shuttle vector for cloning of genes in B. subtilis in cases when high gene dosage might be a problem.

Bacillus subtilis↗
PubMed1987

Isolation and characterization of forespores from Bacillus megaterium.

A procedure for isolation of intact forespores from sporulating Bacillus megaterium cells was developed. The cells were digested with lysozyme and made to release free forespores from the protoplasts by disruption of the cytoplasmic membrane with sonication in phosphate buffer containing 10% glycerol. The suitability of the procedure was confirmed by recovery of dipicolinic acid in the isolated forespores and an electron microscopic observation. The fine structure of the forespores prepared at 6 hr (t6) after initiation of sporulation was similar to that of mature spores, except that the cortex layer and primordial cell wall were thinner and the core was larger. The density, determined by density gradient centrifugation, of the forespores isolated at t6, t10, t12, and mature spores was estimated to be 1.2783, 1.2875, 1.2861, and 1.2858, respectively. The isolated forespores at t6 and t8 were extremely heat labile (D80 of 9.5 and 21.5 min, respectively) relative to mature spores (D80 of 277.8 min). These forespores were also less resistant to organic solvents. Germination of the forespores as well as mature spores was induced by KNO3, D-glucose, and L-leucine. Forespores at t6 were more sensitive to KNO3-induced germination than those at t10, t12, and mature spores when measured by reduction in the optical density of cell suspension.

Bacillus megaterium↗
PubMed1987

Effects of temperature-sensitive variants of the Bacillus subtilis dnaB gene on the replication of a low-copy-number plasmid.

The dnaB gene of Bacillus subtilis is involved in the initiation of DNA replication and also in the binding of the chromosomal origin to the bacterial membrane. We studied the effect of temperature-sensitive dnaB mutants (dnaB1 and dnaB19) on the replication and on the DNA-membrane binding of the plasmid pKW1, which was derived from the low-copy-number plasmid pBS2. In the dnaB19 mutant, pKW1 was not able to replicate at the restrictive temperature. In the dnaB1 mutant, however, the dimeric form of pKW1 DNA was preferentially produced as the restrictive temperature, but the replication of the monomeric form was totally blocked. We also examined the effects of the dnaB(Ts) gene on the DNA-membrane binding of both the double-stranded and single-stranded DNA from pKW1. The single-stranded DNA from pKW1 was prepared from the DNA of the phage M13 mp19, which contained the origin of replication of pKW1. In the dnaB1 mutant, pKW1 DNA in both the double-stranded and single-stranded form was released from the membrane at the restrictive temperature. On the other hand, in the dnaB19 mutant, only double-stranded DNA, and not single-stranded DNA, was released from the membrane at the restrictive temperature. These results suggest that the product of the dnaB gene has at least two domains which influence the replication of DNA and the binding of DNA to the cell membrane in separate ways.

Bacillus subtilis↗
PubMed1986

Cross-reaction of synthetic alpha-(1----3)-branched glucans with rabbit anti-N4 dextran.

Cross-reactions of four synthetic branched glucans (3-O-alpha-D-glucopyranosyl-(1----6)-alpha-D-glucopyranans: V39, V17, V37, and V32, each containing one unit glucose branches amounting to 11-12%, 33-43%, 50-54%, and 71-100%, respectively) with rabbit anti-N4 dextran were examined. All four samples precipitated antibodies raised in rabbits by injecting N4 dextran-concanavalin A conjugate. The ability of glucans to precipitate antibody depended on the quantity of branches, samples with more branches precipitating less antibody nitrogen under the same conditions. This may indicate an inhibitory effect of the branches on precipitation. Oligosaccharide inhibition assay showed that the precipitation reactions were specific for (1----6)-alpha-D-glucopyranosyl linkages, and the maximum size of the alpha-(1----6)-specific antibody combining site corresponded to isomaltopentaose. Determination of antibody nitrogen and glucan in the precipitates indicated that the ratios of one combining site of antibody to numbers of glucose residues were 1:9 (V39), 1:11 (V17), and 1:16 (V37) in the extreme antibody excess region. A synthetic sample of manno-glucan ((1----6)-alpha-D-glucopyranan containing about 27% of randomly linked 3-O-alpha-D-mannopyranosyl side chains) also reacted with the same antibody.

Animals↗